Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H||WB, FC, ICC, ELISA||M||Purified||Monoclonal Antibody|
|Presentation||Purified Ascites presented as a liquid in PBS with 0.1% sodium azide.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at from date of receipt.|
|Material Size||100 µg|
|Anti-Oct-4, clone 10H11.2||2474968|
|Anti-Oct-4, clone 10H11.2 - 2392284||2392284|
|Anti-Oct-4, clone 10H11.2 - 1991253||1991253|
|Anti-Oct-4, clone 10H11.2 - 2066310||2066310|
|Anti-Oct-4, clone 10H11.2 - LV1679970||LV1679970|
|Anti-Oct-4, clone 10H11.2 - LV1786447||LV1786447|
|Anti-Oct-4, clone 10H11.2 - NG1865606||NG1865606|
|Anti-Oct-4, clone 10H11.2 - NG1927045||NG1927045|
|Anti-Oct-4, clone 10H11.2 - PSO1553824||PSO1553824|
|Anti-Oct-4, clone 10H11.2 -2512486||2512486|
|Anti-Oct-4, clone 10H11.2 -2580657||2580657|
|Anti-Oct-4, clone 10H11.2 -2610382||2610382|
|Anti-Oct-4, clone 10H11.2 -2683913||2683913|
|Anti-Oct-4, clone 10H11.2 -2697574||2697574|
|Anti-Oct-4, clone 10H11.2 -2727265||2727265|
References | 18 Available | See All References
|Reference overview||Application||Pub Med ID|
|Transcriptional profiling of ectoderm specification to keratinocyte fate in human embryonic stem cells. |
Tadeu, AM; Lin, S; Hou, L; Chung, L; Zhong, M; Zhao, H; Horsley, V
PloS one 10 e0122493 2015
In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ-secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions.
|Progerin expression disrupts critical adult stem cell functions involved in tissue repair. |
Pacheco, LM; Gomez, LA; Dias, J; Ziebarth, NM; Howard, GA; Schiller, PC
Aging 6 1049-63 2014
Vascular disease is one of the leading causes of death worldwide. Vascular repair, essential for tissue maintenance, is critically reduced during vascular disease and aging. Efficient vascular repair requires functional adult stem cells unimpaired by aging or mutation. One protein candidate for reducing stem cell?mediated vascular repair is progerin, an alternative splice variant of lamin A. Progerin results from erroneous activation of cryptic splice sites within the LMNA gene, and significantly increases during aging. Mutations triggering progerin overexpression cause the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS), in which patients die at approximately 13-years of age due to atherosclerosis-induced disease. Progerin expression affects tissues rich in cells that can be derived from marrow stromal cells (MSCs. Studies using various MSC subpopulations and models have led to discrepant results. Using a well-defined, immature subpopulation of MSCs, Marrow Isolated Adult Multilineage Inducible (MIAMI) cells, we find progerin significantly disrupts expression and localization of self-renewal markers, proliferation, migration, and membrane elasticity. One potential treatment, farnesyltransferase inhibitor, ameliorates some of these effects. Our results confirm proposed progerin-induced mechanisms and suggest novel ways in which progerin disturbs critical stem cell functions collectively required for proper tissue repair, offering promising treatment targets for future therapies.
|Metabolic profiling and flux analysis of MEL-2 human embryonic stem cells during exponential growth at physiological and atmospheric oxygen concentrations. |
Turner, J; Quek, LE; Titmarsh, D; Krömer, JO; Kao, LP; Nielsen, L; Wolvetang, E; Cooper-White, J
PloS one 9 e112757 2014
As human embryonic stem cells (hESCs) steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirements of hESCs, precluding the rational design and optimisation of such platforms. In this study, we undertook an in-depth characterisation of MEL-2 hESC metabolic behaviour during the exponential growth phase, combining metabolic profiling and flux analysis tools at physiological (hypoxic) and atmospheric (normoxic) oxygen concentrations. To overcome variability in growth profiles and the problem of closing mass balances in a complex environment, we developed protocols to accurately measure uptake and production rates of metabolites, cell density, growth rate and biomass composition, and designed a metabolic flux analysis model for estimating internal rates. hESCs are commonly considered to be highly glycolytic with inactive or immature mitochondria, however, whilst the results of this study confirmed that glycolysis is indeed highly active, we show that at least in MEL-2 hESC, it is supported by the use of oxidative phosphorylation within the mitochondria utilising carbon sources, such as glutamine to maximise ATP production. Under both conditions, glycolysis was disconnected from the mitochondria with all of the glucose being converted to lactate. No difference in the growth rates of cells cultured under physiological or atmospheric oxygen concentrations was observed nor did this cause differences in fluxes through the majority of the internal metabolic pathways associated with biogenesis. These results suggest that hESCs display the conventional Warburg effect, with high aerobic activity despite high lactate production, challenging the idea of an anaerobic metabolism with low mitochondrial activity. The results of this study provide new insight that can be used in rational bioreactor design and in the development of novel culture media for hESC maintenance and expansion.
|Generation of iPS Cells from Human Hair Follice Dermal Papilla Cells. |
Muchkaeva, IA; Dashinimaev, EB; Artyuhov, AS; Myagkova, EP; Vorotelyak, EA; Yegorov, YY; Vishnyakova, KS; Kravchenko, IE; Chumakov, PM; Terskikh, VV; Vasiliev, AV
Acta naturae 6 45-53 2014
Dermal papilla (DP) cells are unique regional stem cells of the skin that induce formation of a hair follicle and its regeneration cycle. DP are multipotent stem cells; therefore we supposed that the efficiency of DPC reprogramming could exceed that of dermal fibroblasts reprogramming. We generated induced pluripotent stem cells from human DP cells using lentiviral transfection with Oct4, Sox2, Klf4, and c-Myc, and cultivation of cells both in a medium supplemented with valproic acid and at a physiological level of oxygen (5%). The efficiency of DP cells reprogramming was ~0.03%, while the efficiency of dermal fibroblast reprogramming under the same conditions was ~0.01%. Therefore, we demonstrated the suitability of DP cells as an alternative source of iPS cells.
|Characterization of embryonic stem-like cells derived from HEK293T cells through miR302/367 expression and their potentiality to differentiate into germ-like cells. |
Wang, L; Zhu, H; Wu, J; Li, N; Hua, J
Cytotechnology 66 729-40 2014
Human induced pluripotent stem (iPS) cells have great value for regenerative medicine, but are facing problems of low efficiency. MicroRNAs are a recently discovered class of 19-25 nt small RNAs that negatively target mRNAs. miR302/367 cluster has been demonstrated to reprogram mouse and human somatic cells to iPS cells without exogenous transcription factors, however, the repetition and differentiation potentiality of miR302/367-induced pluripotent stem (mirPS) cells need to be improved. Here, we showed overexpression of miR302/367 cluster reprogrammed human embryonic kidney 293T cells into mirPS cells in serum-free N2B27-based medium. The mirPS cells had similar morphology with embryonic stem cells, and expressed pluripotent markers including Oct4, Sox2, Klf4, and Nanog. In addition, through formation of embryoid bodies, various cells and tissues from three germ layers could be determined. Moreover, we examined the potential of mirPS cells differentiating into germ cells both in vitro and in vivo. Taken together, these data might provide a new source of cells and technique for the investigation of the mechanisms underlying reprogramming and pluripotency.
|Identification of rabbit annulus fibrosus-derived stem cells. |
Liu, C; Guo, Q; Li, J; Wang, S; Wang, Y; Li, B; Yang, H
PloS one 9 e108239 2014
Annulus fibrosus (AF) injuries can lead to substantial deterioration of intervertebral disc (IVD) which characterizes degenerative disc disease (DDD). However, treatments for AF repair/regeneration remain challenging due to the intrinsic heterogeneity of AF tissue at cellular, biochemical, and biomechanical levels. In this study, we isolated and characterized a sub-population of cells from rabbit AF tissue which formed colonies in vitro and could self-renew. These cells showed gene expression of typical surface antigen molecules characterizing mesenchymal stem cells (MSCs), including CD29, CD44, and CD166. Meanwhile, they did not express negative markers of MSCs such as CD4, CD8, and CD14. They also expressed Oct-4, nucleostemin, and SSEA-4 proteins. Upon induced differentiation they showed typical osteogenesis, chondrogenesis, and adipogenesis potential. Together, these AF-derived colony-forming cells possessed clonogenicity, self-renewal, and multi-potential differentiation capability, the three criteria characterizing MSCs. Such AF-derived stem cells may potentially be an ideal candidate for DDD treatments using cell therapies or tissue engineering approaches.
|Synergistic effect of trichostatin A and scriptaid on the development of cloned rabbit embryos. |
Chen, CH; Du, F; Xu, J; Chang, WF; Liu, CC; Su, HY; Lin, TA; Ju, JC; Cheng, WT; Wu, SC; Chen, YE; Sung, LY
Theriogenology 79 1284-93 2013
The first successful rabbit SCNT was achieved more than one decade ago, yet rabbits remain one of the most difficult species to clone. The present study was designed to evaluate the effects of two histone deacetylase inhibitors (HDACis), namely trichostatin A (TSA) and scriptaid (SCP), on cloning efficiency in rabbits. The in vitro development, acetylation levels of histone H4 lysine 5 (H4K5), and octamer-binding transcription factor 4 (Oct-4) expression patterns of cloned embryos were systemically examined after various HDACi treatments. Supplementation of TSA (50 nM) or SCP (250 nM) in the culture medium for 6 hours improved blastocyst development rates of cloned embryos compared with the treatment without HDACi. The combined treatment with TSA (50 nM) and SCP (250 nM) further enhanced morula (58.6%) and blastocyst (49.4%) rates in vitro. More importantly, compared with single HDACi treatments, embryos with the combined treatment had a higher level of H4K5 and an increased total cell number (203.7 ± 14.4 vs. 158.9 ± 9.0 or 162.1 ± 8.2; P less than 0.05) with a better Oct-4 expression pattern in hatching blastocysts, indicating substantially improved embryo quality. This was apparently the first report regarding Oct-4 expression in cloned rabbit embryos. We inferred that most cloned rabbit embryos had an aberrant inner cell mass (ICM) structure accompanied with abnormal spatial distribution of Oct-4 signals. This study demonstrated a synergistic effect of TSA and SCP treatments on cloned rabbit embryos, which might be useful to improve cloning efficiency in rabbits.
|High-content imaging-based screening of microenvironment-induced changes to stem cells. |
Vega, SL; Liu, E; Patel, PJ; Kulesa, AB; Carlson, AL; Ma, Y; Becker, ML; Moghe, PV
Journal of biomolecular screening 17 1151-62 2012
Effective screening methodologies for cells are challenged by the divergent and heterogeneous nature of phenotypes inherent to stem cell cultures, particularly on engineered biomaterial surfaces. In this study, we showcase a high-content, confocal imaging-based methodology to parse single-cell phenotypes by quantifying organizational signatures of specific subcellular reporter proteins and applied this profiling approach to three human stem cell types (embryonic-human embryonic stem cell [hESC], induced pluripotent-induced pluripotent stem cell [iPSC], and mesenchymal-human mesenchymal stem cell [hMSC]). We demonstrate that this method could distinguish self-renewing subpopulations of hESCs and iPSCs from heterogeneous populations. This technique can also provide insights into how incremental changes in biomaterial properties, both physiochemical and mechanical, influence stem cell fates by parsing the organization of stem cell proteins. For example, hMSCs cultured on polymeric films with varying degrees of poly(ethylene glycol) to modulate osteogenic differentiation were parsed using high-content organization of the cytoskeletal protein F-actin. In addition, hMSCs cultured on a self-assembled monolayer platform featuring compositional gradients were screened and descriptors obtained to correlate substrate variations with adipogenic lineage commitment. Taken together, high-content imaging of structurally sensitive proteins can be used as a tool to identify stem cell phenotypes at the single-cell level across a diverse range of culture conditions and microenvironments.
|Dynamic profiles of Oct-4, Cdx-2 and acetylated H4K5 in in-vivo-derived rabbit embryos. |
Chen, CH; Xu, J; Chang, WF; Liu, CC; Su, HY; Chen, YE; Du, F; Sung, LY
Reproductive biomedicine online 25 358-70 2012
This study documents the spatial and temporal distribution of Oct-4, Cdx-2 and acetylated H4K5 (H4K5ac) by immunocytochemistry staining using in-vivo-derived rabbit embryos at different stages: day-3 compact morulae, day-4 early blastocysts, day-4 expanded blastocysts, day-5 blastocysts, day-6 blastocysts and day-7 blastocysts. The Oct-4 signal was stronger in the inner cell mass (ICM)/epiblast cells than in the trophectoderm (TE) cells in all blastocyst stages except day-4 expanded blastocysts, where the signal was similarly weak in both the ICM and TE cells. The Cdx-2 signal was first detected in a small number of TE cells of day-4 early blastocysts, and became evident in the TE cells exclusively afterwards. A consistently strong H4K5ac signal was observed in the TE cells in all blastocyst stages examined. In particular, this signal was stronger in the TE than in the ICM cells in day-4 early blastocysts, day-4 expanded blastocysts and day-5 blastocysts. Double staining of H4K5ac with either Oct-4 or Cdx-2 on embryos at different blastocyst stages confirmed these findings. This work suggests that day 4 is a critical timing for lineage formation in rabbit embryos. A combination of Oct-4, Cdx-2 and H4K5ac can be used as biomarkers to identify different lineage cells in rabbit blastocysts.
|Spatial and temporal distribution of Oct-4 and acetylated H4K5 in rabbit embryos. |
Chen, CH; Chang, WF; Liu, CC; Su, HY; Shyue, SK; Cheng, WT; Chen, YE; Wu, SC; Du, F; Sung, LY; Xu, J
Reproductive biomedicine online 24 433-42 2012
Rabbit is a unique species to study human embryology; however, there are limited reports on the key transcription factors and epigenetic events of rabbit embryos. This study examined the Oct-4 and acetylated H4K5 (H4K5ac) patterns in rabbit embryos using immunochemistry staining. The average intensity of the Oct-4 signal in the nuclei of the whole embryo spiked upon fertilization, then decreased until the 8-cell stage and increased afterwards until the compact morula (CM) stage. It decreased thereafter from the CM stage to the early blastocyst (EB) stage, with a minimum at the expanded blastocyst (EXPB) stage and came back to a level similar to that of the CM-stage embryos in the hatching blastocysts (HB). The Oct-4 signal was observed in both the inner cell mass (ICM) and the trophectoderm (TE) cells of blastocysts. The average H4K5ac signal intensity of the whole embryo increased upon fertilization, started to decrease at the 4-cell stage, reached a minimum at the 8-cell stage, increased again at the EXPB stage and peaked at the HB stage. While TE cells maintained similar levels of H4K5ac throughout the blastocyst stages, ICM cells of HB showed higher levels of H4K5ac than those of EB and EXPB. Understanding key genetic and epigenetic events during early embryo development will help to identify factors contributing to embryo losses and consequently improve embryo survival rates. As a preferred laboratory species for many human disease studies such as atherosclerosis, rabbit is also a pioneer species in the development of several embryo biotechnologies, such as IVF, transgenesis, animal cloning, embryo cryopreservation and embryonic stem cells. However, there are limited reports on key transcription factors and epigenetic events of rabbit embryos. In the present study, we documented the temporal and spatial distribution of Oct-4 protein and H4K5 acetylation during early embryo development using the immunostaining approach. We also compared the patterns of these two important biomarkers between the inner cell mass (ICM) and the trophectoderm (TE) cells in blastocyst-stage embryos. Our findings suggest that a combination of Oct-4, H4K5ac and possibly other biomarkers such as Cdx-2 is needed to accurately identify different lineages of cells in morula and blastocyst stage rabbit embryos. Importantly, we revealed a novel wave of Oct-4 intensity change in the ICM cells of rabbit blastocysts. The signal was high at the early blastocyst stage, reached a minimum at the expanded blastocyst stage and returned to a high level at the hatching blastocyst stage. We hypothesize that the signal may have reflected the regulation of Oct-4 through enhancer switching and therefore may be related to cell lineage formation in rabbit embryos. These findings enrich our understanding on key genetic and epigenetic programming events during early embryo development in rabbits.
|Oct-4B isoform is differentially expressed in breast cancer cells: hypermethylation of regulatory elements of Oct-4A suggests an alternative promoter and transcriptional start site for Oct-4B transcription. |
Wang, Yajuan, et al.
Biosci. Rep., 31: 109-15 (2011) 2011
The human Oct-4 gene has three isoforms, Oct-4A, Oct-4B and Oct-4B1, which are thought to be derived from alternative splicing. It remains controversial whether the Oct-4 gene is expressed in cancer cells. Expression of Oct-4A is regulated by two elements, the PE (proximal enhancer) and DE (distal enhancer), but the expression and regulation of Oct-4B are not well known. Here, we firstly report that Oct-4B is expressed at low levels in MCF-7 cells, while the Oct-4A gene is inactivated. By analysing the function of different promoter constructs and the DNA methylation status of three regulatory regions, we demonstrate that the Oct-4A gene in MCF-7 cells is repressed by epigenetic control rather than transcriptional control. In addition, we speculate that the transcription of Oct-4B in MCF-7 cells is differentially regulated by additional regulatory elements. This work will enhance the understanding of Oct-4 gene in differential regulation.
|Characterization of differential properties of rabbit tendon stem cells and tenocytes. |
Zhang, J; Wang, JH
BMC musculoskeletal disorders 11 10 2010
Tendons are traditionally thought to consist of tenocytes only, the resident cells of tendons; however, a recent study has demonstrated that human and mouse tendons also contain stem cells, referred to as tendon stem/progenitor cells (TSCs). However, the differential properties of TSCs and tenocytes remain largely undefined. This study aims to characterize the properties of these tendon cells derived from rabbits.TSCs and tenocytes were isolated from patellar and Achilles tendons of rabbits. The differentiation potential and cell marker expression of the two types of cells were examined using histochemical, immunohistochemical, and qRT-PCR analysis as well as in vivo implantation. In addition, morphology, colony formation, and proliferation of TSCs and tenocytes were also compared.It was found that TSCs were able to differentiate into adipocytes, chondrocytes, and osteocytes in vitro, and form tendon-like, cartilage-like, and bone-like tissues in vivo. In contrast, tenocytes had little such differentiation potential. Moreover, TSCs expressed the stem cell markers Oct-4, SSEA-4, and nucleostemin, whereas tenocytes expressed none of these markers. Morphologically, TSCs possessed smaller cell bodies and larger nuclei than ordinary tenocytes and had cobblestone-like morphology in confluent culture whereas tenocytes were highly elongated. TSCs also proliferated more quickly than tenocytes in culture. Additionally, TSCs from patellar tendons formed more numerous and larger colonies and proliferated more rapidly than TSCs from Achilles tendons.TSCs exhibit distinct properties compared to tenocytes, including differences in cell marker expression, proliferative and differentiation potential, and cell morphology in culture. Future research should investigate the mechanobiology of TSCs and explore the possibility of using TSCs to more effectively repair or regenerate injured tendons.Full Text Article
|Efficient derivation of embryonic stem cells from nuclear transfer and parthenogenetic embryos derived from cryopreserved oocytes. |
Sung LY, Chang CC, Amano T, Lin CJ, Amano M, Treaster SB, Xu J, Chang WF, Nagy ZP, Yang X, Tian XC
Cell Reprogram 12 203-11. 2010
Deriving histocompatible embryonic stem (ES) cells by somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA) requires fresh oocytes, which prevents their applications in humans. Here, we evaluated the efficiency of deriving ES cells from mature metaphase II (MII) and immature metaphase I (MI) vitrified oocytes, by PA or SCNT, in a mouse model. We successfully generated ES cell lines from PA (MII and MI) and SCNT (MII and MI) blastocysts. These cell lines expressed genes and antigens characteristic of pluripotent ES cells and produced full-term pups upon tetraploid embryo complementation. This study established an animal model for efficient generation of patient-specific ES cell lines using cryopreserved oocytes. This is a major step forward in the application of therapeutic cloning and parthenogenetic technology in human regenerative medicine and will serve as an important alternative to the iPS cell technology in countries/regions where these technologies are permitted.
|Efficient, high-throughput transfection of human embryonic stem cells. |
Moore, JC; Atze, K; Yeung, PL; Toro-Ramos, AJ; Camarillo, C; Thompson, K; Ricupero, CL; Brenneman, MA; Cohen, RI; Hart, RP
Stem cell research & therapy 1 23 2010
Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications.We investigated the ability of two commercially available electroporation systems, the Nucleofection® 96-well Shuttle® System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency.Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However, the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection® 96-well Shuttle® System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency, producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally, we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference.Our results indicate that these electroporation methods provide a reliable, efficient, and high-throughput approach to the genetic manipulation of hESC.
|Ago2 immunoprecipitation identifies predicted microRNAs in human embryonic stem cells and neural precursors. |
Goff, LA; Davila, J; Swerdel, MR; Moore, JC; Cohen, RI; Wu, H; Sun, YE; Hart, RP
PloS one 4 e7192 2009
MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation.SOLiD ultra-deep sequencing identified greater than 10(7) unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs.Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation.Full Text Article
|The intracellular distribution of the ES cell totipotent markers OCT4 and Sox2 in adult stem cells differs dramatically according to commercial antibody used. |
Patricia A Zuk, Patricia A Zuk
Journal of cellular biochemistry 106 867-77 2009
To characterize ES cells, researchers have at their disposal a list of pluripotent markers, such as OCT4. In their quest to determine if adult stem cell populations, such as MSCs and ASCs, are pluripotent, several groups have begun to report the expression of these markers in these cells. Consistent with this, human ASCs (hASCs) are shown in this study to express a plethora of ES pluripotent markers at the gene and protein level, including OCT4, Sox2, and Nanog. When intracellular distribution is examined in hASCs, both OCT4 and Sox2 are expressed within the nuclei of hASCs, consistent with their expression patterns in ES cells. However, a significant amount of expression can be noted within the hASC cytoplasm and a complete absence of nuclear expression is observed for Nanog. Recent descriptions of OCT4 transcript variants may explain the cytoplasmic expression of OCT4 in hASCs and consistent with this, hASCs do express both the OCT4A and 4B transcript variants at the gene level. However, discrepancies arise when these three pluripotent markers are studied at the protein level. Specifically, distinct differences in intracellular expression patterns were noted for OCT4, Sox2, and Nanog from commercial antibody to commercial antibody. These antibody discrepancies persisted when hMSCs and rat ASCs and MSCs were examined. Therefore, confirming the expression of OCT4, Sox2, and Nanog in adult stem cells with today's commercial antibodies must be carefully considered before the designation of pluripotent can be granted.
|Targeting cancer stem cells through L1CAM suppresses glioma growth. |
Bao, S; Wu, Q; Li, Z; Sathornsumetee, S; Wang, H; McLendon, RE; Hjelmeland, AB; Rich, JN
Cancer research 68 6043-8 2008
Malignant gliomas are lethal cancers that display striking cellular heterogeneity. A highly tumorigenic glioma tumor subpopulation, termed cancer stem cells or tumor-initiating cells, promotes therapeutic resistance and tumor angiogenesis. Therefore, targeting cancer stem cells may improve patient survival. We interrogated the role of a neuronal cell adhesion molecule, L1CAM, in glioma stem cells as L1CAM regulates brain development and is expressed in gliomas. L1CAM(+) and CD133(+) cells cosegregated in gliomas, and levels of L1CAM were higher in CD133(+) glioma cells than normal neural progenitors. Targeting L1CAM using lentiviral-mediated short hairpin RNA (shRNA) interference in CD133(+) glioma cells potently disrupted neurosphere formation, induced apoptosis, and inhibited growth specifically in glioma stem cells. We identified a novel mechanism for L1CAM regulation of cell survival as L1CAM knockdown decreased expression of the basic helix-loop-helix transcription factor Olig2 and up-regulated the p21(WAF1/CIP1) tumor suppressor in CD133(+) glioma cells. To determine if targeting L1CAM was sufficient to reduce glioma stem cell tumor growth in vivo, we targeted L1CAM in glioma cells before injection into immunocompromised mice or directly in established tumors. In each glioma xenograft model, shRNA targeting of L1CAM expression in vivo suppressed tumor growth and increased the survival of tumor-bearing animals. Together, these data show that L1CAM is required for maintaining the growth and survival of CD133(+) glioma cells both in vitro and in vivo, and L1CAM may represent a cancer stem cell-specific therapeutic target for improving the treatment of malignant gliomas and other brain tumors.
|Microporous membrane growth substrates for embryonic stem cell culture and differentiation. |
Steven D Sheridan,Sonia Gil,Matthew Wilgo,Aldo Pitt
Methods in cell biology 86 2008
As the field of embryonic stem cell culture and differentiation advances, many diverse culturing techniques will ultimately be necessary in order to fully reproduce the various environments these cells normally encounter during development. Although most of the work to date has been performed on solid plastic supports, this growth support has several limitations in its representation of the in vivo environment. Impermeable substrates force the cells to exchange their gas and nutrients exclusively through the top side of the cultured cells. In contrast, cells growing in vivo are exposed from several directions to factors from the blood, other cells, soluble factors, and liquid-air interfaces. Additionally, solid plastic presents a smooth two-dimensional surface that is not experienced in vivo. Therefore, the use of traditional plastic presents limitations upon normal cellular morphology, function, and differentiation. An important alternative to growth on solid plastic is the growth of cells on microporous membranes. One of the many advantages to cell growth on porous membrane substrates is their ability to provide a surface that better mimics a three-dimensional in vivo setting. A porous membrane allows multidirectional exposure to nutrients and waste products. In addition, the membrane separation of dual chambers allows for the coculture of cells of different origin to study how cells interact through indirect signaling or through providing a conditioned niche for the proper growth and differentiation of cell types.
|Human Stem Cell Systems|
|Anti-Oct-4, clone 10H11.2 - Data Sheet|
|STEMCCA Lentivirus Reprogramming Kits|
|Efficient generation of transgene-free human and mouse iPS cells using a cell-permeant Tat-Cre protein|